Tannic Acid Solution: A Better Fixative Solution Than Formalin for Elastin and Collagen-Toxic and Morphological Assessment.

Formaldehyde is commonly used worldwide, even though it is classified as carcinogenic to humans by the International Agency for Research on Cancer. This has motivated intensive investigations of formaldehyde substitutes, and recently, some alternative solutions were found, which can potentially replace it. Previous research showed that tannic acid (TA) in glutaraldehyde solution has the ability to stabilize elastin and collagen. This provided a basis for the development of a new alcoholic fixative solution, particularly aimed at extracellular matrix components, with TA as a main component. Heart, brain, and intestinal samples were fixed by immersion in 10% regular formalin solution (RFS), 70% ethanol solution (ES), and tannic acid ethanolic solution (TAES). Next, tissue fragments were prepared for routine histology procedures. The toxicity of TA was analyzed using in silico tests for mutagenicity, as well as for cutaneous and respiratory toxicity. Analyses of photomicrographs demonstrated that all fixative solutions have the ability to preserve the fragments. The quantitative analyses showed that capability of TAES to preserve and stabilize elastin and collagen is superior to that of RFS and ES. We demonstrated that TA is not mutagenic, and it is less toxic for skin and respiratory tract. We therefore conclude that TAES can potentially represent a powerful and feasible alternative solution for fixing extracellular matrix of microscopic examination samples. Anat Rec, 301:1544-1550, 2018. © 2018 Wiley Periodicals, Inc.

Toxicity of Senna occidentalis seeds in laying hens and its effects on egg production.

Senna occidentalis is a toxic leguminous plant found in many tropical and subtropical regions of the world and causes poisoning mainly in confined animals. The seeds are the most toxic part of the plant and may be present in animal rations. The main toxic component of the S. occidentalis seed is a dianthrone, an anthraquinone-derived compound that affects mitochondrial function. This study evaluated the effects on egg production of low-level contamination of the S. occidentalis in the laying hens' diet. Forty-eight one-day-old pullets were randomly allocated into two treatment groups: control, birds that received no experimental treatment; and external and internal tegument (ET/IT), birds that received a diet containing 0.2% of ET/IT of S. occidentalis seeds throughout their life cycle (42 weeks). The birds were monitored for clinical signs of poisoning, and the production and quality of eggs were recorded. Necropsies were conducted at the end of the experimental period. None of the layers showed any clinical signs of poisoning, decreases in feed intake or alterations of the body weight gain. A marked reduction in egg production and, consequently, a lower feed efficiency in ET/IT group were measured. Ovaries were the most affected organ in birds from the ET/IT group, and yolk leaking and dysplasia of the inner layer of the vitelline membrane were observed. S. occidentalis was shown to be toxic for laying hens. Considering these results, it is feasible to assume that the constant presence of low concentrations of S. occidentalis seeds in rations represents a threat to the poultry industry.

Maternal Ingestion of Ipomoea carnea: Effects on Goat-Kid Bonding and Behavior.

Ipomoea carnea is a toxic plant found in Brazil and other tropical and subtropical countries and often causes poisoning of livestock. The plant contains the alkaloids swainsonine and calystegines, which inhibit key cellular enzymes and cause systematic cell death. This study evaluated the behavioral effects of prenatal ingestion of this plant on dams and their kids. Twenty-four pregnant goats were randomly allocated into four treatment groups and received the following doses (g/kg BW) of fresh I. carnea: 0 (control group), 1.0 (IC1), 3.0 (IC3), and 5.0 (IC5) from day 27 of gestation until parturition. Dam and kid bonding and behavior were evaluated by several tests, immediately after birth until six weeks of age. Dams from IC3 and IC5 groups spent less time paying attention to the newborn. There was a lack of maternal-infant bonding due to I. carnea intoxication. Kids from treated dams had difficulty in standing, suckling, and in recognizing their mother hours after birth. I. carnea can also compromise the kids' ability to learn and to retain spatial memory. We suggest that kids from pregnant goats given I. carnea during gestation have significant behavioral alterations and developmental delays that may compromise their survival.

Intoxication by Cyanide in Pregnant Sows: Prenatal and Postnatal Evaluation.

Cyanide is a ubiquitous chemical in the environment and has been associated with many intoxication episodes; however, little is known about its potentially toxic effects on development. The aim of this study was to evaluate the effects of maternal exposure to potassium cyanide (KCN) during pregnancy on both sows and their offspring. Twenty-four pregnant sows were allocated into four groups that orally received different doses of KCN (0.0, 2.0, 4.0, and 6.0 mg/kg of body weight) from day 21 of pregnancy to term. The KCN-treated sows showed histological lesions in the CNS, thyroid follicle enlargement, thyroid epithelial thickening, colloid reabsorption changes, and vacuolar degeneration of the renal tubular epithelium. Sows treated with 4.0 mg/kg KCN showed an increase in the number of dead piglets at birth. Weaned piglets from all KCN-treated groups showed histological lesions in the thyroid glands with features similar to those found in their mothers. The exposure of pregnant sows to cyanide thus caused toxic effects in both mothers and piglets. We suggest that swine can serve as a useful animal model to assess the neurological, goitrogenic, and reproductive effects of cyanide toxicosis.

Immunosuppressive effects of Pteridium aquilinum enhance susceptibility to urethane-induced lung carcinogenesis.

Pteridium aquilinum (bracken fern), one of the most important toxic plants in the world, contains the toxic norsequiterpene ptaquiloside that induces cancers in humans and farm animals. Previous studies in the laboratory demonstrated immunotoxic effects produced by ptaquiloside, which are characterized by suppression of natural killer (NK) cell activity (i.e. cytotoxicity and interferon [IFN]-γ production). However, it is unknown whether these immunosuppressive effects could contribute to carcinogenesis in situ in general because of the important function of NK cells in innate killing of tumor cells. This study assessed the impact of P. aquilinum-induced immunosuppression on urethane-induced lung cancer in C57BL/6 mice. Adult mice were treated with an extract of P. aquilinum (30 g/kg/day) by gavage once daily for 14 days, followed by gavage (5 days/week) during an 11-week period that was accompanied by treatment with urethane (1 g/kg) via once-weekly intraperitoneal injection; 20 weeks after the end of the treatment period, all lungs were evaluated. The results indicated there was a significant increase in lung nodule number as well as in multiplicity of lesions in mice treated with both P. aquilinum and urethane (PU group) compared to values in mice treated only with the urethane (U group). In addition, histologic evaluation revealed a 76% increase in the rate of lung adenomas and a 41% increase in rate of bronchiolization of alveoli in the mice from the PU group compared to levels seen in mice within the U group. Taken together, the results here show for the first time that immunosuppressive effects of P. aquilinum could increase the risk of cancer formation in exposed hosts.

Zearalenone, an estrogenic mycotoxin, is an immunotoxic compound.

The aim of this study was to assess the toxic effects of zearalenone (ZEA) on the immune function. Ovariectomised rats were treated daily by gavage with 3.0 mg/kg of ZEA for 28 days. Body weight gain, food consumption, haemotological parameters, lymphoid organs, and their cellularities were evaluated. Moreover, acquired immune responses and macrophage activity were also assessed. ZEA promoted reduction in body weight gain, which is not fully explained by diminished food consumption. Despite no effect on haematological parameters, ZEA caused thymic atrophy with histological and thymocyte phenotype changes and decrease in the B cell percentage in the spleen. With respect to acquired and innate immune responses, no statistically significant differences in delayed-type hypersensitivity were noticed; however, in the ZEA-treated rats, antibody production and peroxide release by macrophages were impaired. The observed results could be related to ZEA activity on ERs; thus, ZEA is an immunotoxic compound similar to estrogen and some endocrine disruptors.

Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium.

Pteridium aquilinum, one of the most important poisonous plants in the world, is known to be carcinogenic to animals and humans. Moreover, our previous studies showed that the immunosuppressive effects of ptaquiloside, its main toxic agent, were prevented by selenium in mouse natural killer (NK) cells. We also verified that this immunosuppression facilitated development of cancer. Here, we performed gene expression microarray analysis in splenic NK cells from mice treated for 14 days with ptaquiloside (5.3 mg/kg) and/or selenium (1.3 mg/kg) to identify gene transcripts altered by ptaquiloside that could be linked to the immunosuppression and that would be prevented by selenium. Transcriptome analysis of ptaquiloside samples revealed that 872 transcripts were expressed differentially (fold change>2 and p<0.05), including 77 up-regulated and 795 down-regulated transcripts. Gene ontology analysis mapped these up-regulated transcripts to three main biological processes (cellular ion homeostasis, negative regulation of apoptosis and regulation of transcription). Considering the immunosuppressive effect of ptaquiloside, we hypothesized that two genes involved in cellular ion homeostasis, metallothionein 1 (Mt1) and metallothionein 2 (Mt2), could be implicated because Mt1 and Mt2 are responsible for zinc homeostasis, and a reduction of free intracellular zinc impairs NK functions. We confirm these hypotheses and show increased expression of metallothionein in splenic NK cells and reduction in free intracellular zinc following treatment with ptaquiloside that were completely prevented by selenium co-treatment. These findings could help avoid the higher susceptibility to cancer that is induced by P. aquilinum-mediated immunosuppressive effects.

The use of ultrasonography to study teratogenicity in ruminants: evaluation of Ipomoea carnea in goats.

BACKGROUND: Ipomoea carnea (I. carnea) is a poisonous plant found in Brazil and other tropical countries that often poison livestock. The plant contains the alkaloids calystegines and mainly swainsonine, which inhibit cellular enzymes and cause systematic cell death. The objective of this study was to evaluate the perinatal effects of I. carnea in goats. METHODS: Forty-seven pregnant goats were randomly allocated into 5 treatment groups and given the following doses (g/kg BW) of I. carnea: 0 (IC0), 1.0 (IC1), 3.0 (IC3), 5.0 (IC5) and 7.5 (IC7). The treatment animals were given fresh I. carnea from day 27 of gestation to parturition. Weight gains and serum biochemistry were evaluated. Fetuses were evaluated using ultrasonographic measurements. RESULTS: Goats from the IC7 group showed clinical signs of poisoning. Ultrasound examination revealed that I. carnea feeding in all treatment groups reduced fetal movement compared to the controls. There was an increase in the total number of birth defects (retrognathia and arthrogyposis) in the IC7 and IC5 groups compared to the controls. CONCLUSION: The results show that I. carnea has teratogenic potential in goats. In addition, ultrasounds were useful in evaluating fetotoxicity and teratogenicity.

The immunomodulatory effects of Ipomoea carnea in rats vary depending on life stage.

Ipomoea carnea Jacq. ssp. fistulosa (Mart. Ex Choisy; Convolvulaceae; I. carnea) possesses a toxic component: an indolizidine alkaloid swainsonine (SW) that has immunomodulatory effects due to its inhibition of glycoprotein metabolism. It is also known that SW is excreted into both the amniotic fluid and milk of female rats exposed to I. carnea. Thus, the aim of this study was to determine whether SW exposure, either in utero or from the milk of dams treated with I. carnea, modulates offspring immune function into adulthood. In addition, adult (70 days old) and juvenile rats (21 days old) were exposed to I. carnea in order to evaluate several other immune parameters: lymphoid organs relative weight and cellularity, humoral and cellular immune responses. Offspring exposed to I. carnea during lactation developed rheumatoid arthritis (RA) in adulthood after an immunogenic challenge. In addition, both adult and juvenile rats exposed to I. carnea showed discrepancies in several immune parameters, but did not exhibit any decrease in humoral immune response, which was enhanced at both ages. These findings indicate that SW modulates immune function in adult rats exposed to SW during lactation and in juvenile and adult rats exposed to SW as juveniles and adults, respectively.

Haematological and immunological effects of repeated dose exposure of rats to integerrimine N-oxide from Senecio brasiliensis.

This study is the first in the literature to focus attention on the possible immunotoxic effect of integerrimine N-oxide content in the butanolic residue (BR) of Senecio brasiliensis, a poisonous hepatotoxic plant that contains pyrrolizidine alkaloids (PAs). PAs have been reported as a pasture and food contaminant and as herbal medicine used worldwide and are responsible for poisoning events in livestock and human beings. After the plant extraction, BR extracted from Senecio brasiliensis was found to contain approximately 70% integerrimine N-oxide by elemental and spectral analyses ((1)H and (13)C NMR), which was administered to adult male Wistar Hannover rats at doses of 3, 6 and 9 mg/kg for 28 days. Body weight gain, food consumption, lymphoid organs, neutrophil analysis, humoural immune response, cellular immune response and lymphocyte analysis were evaluated. Our study showed that integerrimine N-oxide could promote an impairment in the body weight gain, interference with blood cell counts and a reducing T cell proliferative activity in rats; however, no differences in the neutrophil activities, lymphocytes phenotyping and humoural and cellular immune responses were observed. It is concluded that doses of integerrimine N-oxide here employed did not produce marked immunotoxic effects.

Intoxication by Senna occidentalis seeds in pregnant goats: prenatal and postnatal evaluation.

Senna occidentalis is a weed toxic to different animal species. Very little is known about the effects of prolonged exposure to low doses of S. occidentalis on developmental toxicology. Thus, the present study proposes an approach to evaluate the perinatal toxicity of S. occidentalis seeds in goats. Twenty-one pregnant goats were fed rations containing 0% (control), 1% (So1 group), 2% (So2 group) and 4% (So4 group) mature S. occidentalis seeds from pregnancy detection on day 27 after mating until parturition; weight gains and serum biochemistry were evaluated. Fetuses were evaluated using ultrasonographic measurements; neonates were evaluated by body morphometry, weight gains, and serum biochemistry. Fetal resorption occurred in 2 So4 dams and one dam died. Only a few minor alterations in serum biochemistry occurred in dams and kids; even so one So4 group dam had tissue lesions as vacuolations in hepatocytes and kidneys; necrosis in skeletal and cardiac muscles and for the first time lesions were observed in sciatic nerve cells. No relevant alterations in body morphometry were observed. This study suggests that 4% S. occidentalis seeds is toxic for pregnant goats, but levels of seeds less than 4% have little impact on fetal and post birth body development.

Low doses of monocrotaline in rats cause diminished bone marrow cellularity and compromised nitric oxide production by peritoneal macrophages.

Monocrotaline (MCT) is a pyrrolizidine alkaloid found in a variety of plants. The main symptoms of MCT toxicosis in livestock are related to hepato- and nephrotoxicity; in rodents and humans, the induction of a pulmonary hypertensive state that progresses to cor pulmonale has received much attention. Although studies have shown that MCT can cause effects on cellular functions that would be critical to those of lymphocytes/macrophages during a normal immune response, no immunotoxicological study on MCT have yet to ever be performed. Thus, the aim of the present study was to evaluate the effect of MCT on different branches of the immune system using the rat--which is known to be sensitive to the effects of MCT--as the model. Rats were treated once a day by gavage with 0.0, 0.3, 1.0, 3.0, or 5.0 mg MCT/kg for 14 days, and then any effects of the alkaloid on lymphoid organs, acquired immune responses, and macrophage activity were evaluated. No alterations in the relative weight of lymphoid organs were observed; however, diminished bone marrow cellularity in rats treated with the alkaloid was observed. MCT did not affect humoral or cellular immune responses. When macrophages were evaluated, treatments with MCT caused no significant alterations in phagocytic function or in hydrogen peroxide (H2O2) production; however, the MCT did cause compromised nitric oxide (NO) release by these cells.

Assessment of the perinatal effects of maternal ingestion of Ipomoea carnea in rats.

It is believed that Ipomoea carnea toxicosis induces abnormal embryogenesis in livestock. Studies on rats treated with I. carnea aqueous fraction (AF) during gestation, revealed litters with decreased body weight, but the characteristic vacuolar lesions promoted by swainsonine, its main toxic principle, were observed only in young rats on postnatal day (PND) 7. However, these alterations could have resulted as consequence of swainsonine placental passage and/or damage or even ingestion of the contaminated milk by pups. Thus, this perinatal work was performed to verify the transplacental passage of swainsonine and its excretion into milk employing the cross-fostering (CF) procedure as a tool of study. Females were treated with AF or vehicle during gestation and after birth pups were fostered between treated and untreated dams. Pup body weight gain (BWG) and histopathology to observe vacuolar degeneration were performed on PND 3 and 7. In addition, swainsonine detection was performed in amniotic fluid and milk from rats treated with the AF during gestation or lactation. BWG was significantly lower only in pups from mothers treated with the plant and fostered to other treated mothers (AF-AF group of pups). The histopathology revealed that pups from treated mothers fostered to untreated ones showed the characteristic vacuolar lesions; however, the lesions from the AF-AF pups were more severe in both periods evaluated. Amniotic fluid and milk analysis revealed the presence of swainsonine excretion into these fluid compartments. Thus, the results from CF and the chemical analysis allowed concluding that swainsonine passes the placental barrier and affects fetal development and milk excretion participates in I. carnea perinatal toxicosis.

The role of alkaloids in Ipomoea carnea toxicosis: a study in rats.

Ipomoea carnea promotes in livestock a toxicosis histologically characterized by vacuolated cells in different organs. The toxic principles of I. carnea are the alkaloids swainsonine and calystegines B1, B2, B3 and Cl. However, it has not been determined whether the effects observed in rats treated with this plant are only due to swainsonine or if the calystegines have some additive toxic effect. Thus, the aim of the present study was to evaluate in rats the toxic effects of the L carnea aqueous fraction (AF) and of its different alkaloids when administered individually at the same concentration as in this fraction, for 14 days. No anorexic effect and/or alteration in body weight was observed in any group. The histopathologic study showed that while calystegines did not produce any toxic effects, swainsonine and I carnea AF promoted vacuolation in different organs, being more severe in the animals from the I. carnea AF group and extensible to other organs evaluated. No alterations were detected in the central nervous system of rats of any group assayed. The results obtained here suggest that calystegines may act as coadjuvants of swainsonine in I carnea toxicosis; however, little can be proposed about the neurotoxic effect of I. carnea since rats did not prove to be a good model for the reproduction of neuronal storage disease.

Prenatal toxicity of cyanide in goats--a model for teratological studies in ruminants.

Although exposure to cyanogenic plants or cyanide during pregnancy has adverse effects, no teratological study with cyanide has been conducted in goats or any other ruminant. The objective of the present study was to evaluate the effects of the maternal exposure to potassium cyanide (KCN) during pregnancy on both dams and offspring and furthermore, to develop a model for prenatal toxicological studies in ruminants. Twenty-six pregnant goats were allocated into four groups and given 0, 1.0, 2.0, or 3.0mg KCN/kg body weight per day orally (administered via twice-daily gavage) from Day 24 of pregnancy to term. However, one control dam and another from the 3.0mg KCN/kg per day group were sacrificed on Day 120. At birth, the kids were examined carefully for gross abnormalities. Three months after birth, the male kids and one dam from each group were sacrificed for histopathological study. Although clinical signs of poisoning were observed in dams, cyanide treatment did not alter the length of gestation or the number of live kids. Two prognata kids were born in the 3.0mg KCN/kg group, and one dam from the same group aborted two fetuses. There were histological lesions only in the KCN-treated dam (and its fetuses) sacrificed on Day 120; these consisted of an increased number of resorption vacuoles of thyroid follicular colloid, and status spongiosis of nervous white matter. This study proposes a new animal model for teratogenic trials that could be important to evaluate the effects of chemicals throughout pregnancy in goats and potentially other ruminants.

Effects of Senna occidentalis on chick bursa of Fabricius.

Senna occidentalis (L) Link (formerly called Cassia occidentalis) is a toxic leguminous plant found ubiquitously as a contaminant of crops. All parts of the plant are toxic, but most of the S. occidentalis toxicity is found in the seeds. S. occidentalis has been shown to be toxic to several animal species, causing degenerative lesions mainly in muscles. This is the first report describing alterations in chick lymphoid organs caused by S. occidentalis seeds. The objectives of this study were to describe the effects of the treatment with seeds and its fraction external tegument (TE) on the development of chicks and their lymphoid organs bursa of Fabricius and spleen. Chicks that received a commercial ration with 1% TE had reduced body and lymphoid organ weights. The bursa of Fabricius presented reduction in the diameters of the follicles, and in the thickness of the cortical and medullary regions. The spleen presented depleted lymphoid tissue in the white pulp. These results indicate that the active principle of S. occidentalis is more concentrated on its TE fraction, and that it can cause weight loss as well as alterations in the lymphoid organs in chicks. The consequences of these alterations should be further investigated.

Alkaloidal components in the poisonous plant, Ipomoea carnea (Convolvulaceae).

Natural intoxication of livestock by the ingestion of Ipomoea carnea (Convolvulaceae) sometimes occurs in tropical regions of the world. Polyhydroxylated alkaloids were isolated from the leaves, flowers, and seeds of the poisonous plant and characterized. Chromatographic separation of the leaf extract resulted in the isolation of swainsonine (1), 2-epi-lentiginosine (2), calystegines B(1) (3), B(2) (4), B(3) (5), and C(1) (6), and N-methyl-trans-4-hydroxy-l-proline (7). The contents of 1 in the fresh leaves and flowers were 0.0029 and 0.0028%, respectively, whereas the contents of 1, 3, and 4 in the seeds were approximately 10 times higher than those in the leaves and flowers. Alkaloids 3, 4, and 6 showed a potent inhibitory activity toward rat lysosomal beta-glucosidase, with IC(50) values of 2.1, 0.75, and 0.84 microM, respectively, and alkaloid 5 was a moderate inhibitor of alpha- and beta-mannosidases. Although alkaloid 1 is known as a powerful inhibitor of lysosomal alpha-mannosidase (IC(50) = 0.02 microM), alkaloid 2, which has been thought to be an intermediate in the biosynthesis of 1, was also a potent inhibitor of alpha-mannosidase with an IC(50) value of 4.6 microM.

Neurotoxicity to pigs and rodents from different fractions of Aeschynomene indica seeds.

Aeschynomene indica seeds cause a vestibulo-cerebellar syndrome in pigs. This experiment studied the toxicity of different plant chemical fractions in pigs to determine a susceptible laboratory species to search for the plant's toxic principle. Hexanic, ethanolic and acetonic extracts of A. indica seeds were administered to 1 pig each. The ethanolic extract killed the experimental pig and 2/4 mice and 0/4 rats. The ethanolic extract was fractionated into ethyl acetate, butanolic. and aqueous remaining residues. The residues were administered by gavage at 0.9 g/kg to groups of 6 mice; those dosed with the ethyl acetate residue developed nervous signs and died. Administrated to 4 pigs, the residue caused clinical signs and histologic lesions similar than those observed in experimental intoxication of swine with A. indica seeds. The active principle of these seeds was in the ethyl acetate residue and mice can be used as an experimental species to test toxicity of substances isolated from this plant.

Toxicokinetics of cyanide in rats, pigs and goats after oral dosing with potassium cyanide.

The aim of the present study was to determine the effect of the species on the toxicokinetics of cyanide and its main metabolite, thiocyanate. Forty-two rats, six pigs and six goats were dosed orally with 3.0 mg KCN/kg body weight, and cyanide and thiocyanate concentrations in blood were measured within 24 h. After the single oral dose, KCN was rapidly absorbed by rats and goats, with a time of peak concentration ( T(max)) of 15 min. The maximum plasma concentration ( C(max)) of cyanide was observed in goats (93.5 micro mol/l), whereas the C(max) of thiocyanate was higher in rats (58.1 micro mol/l). The elimination half-life ( t(1/2)) and volume of distribution ( Vd(area)) of both cyanide and thiocyanate were higher in goats (1.28 and 13.9 h, and 0.41 and 1.76 l/kg, respectively). Whereas the area under the curve (AUC) of cyanide was significantly higher in goats (234.6 micro mol.l/h), the AUC of thiocyanate was higher in rats (846.5 micro mol.l/h). In conclusion, the results of the present study support the hypothesis that the metabolism of cyanide and its main metabolite, thiocyanate, is species-linked, with the goat being more sensitive to the toxic effects of cyanide/thiocyanate.

Milk transfer of cyanide and thiocyanate: Cyanide exposure by lactation in goats.

The present work was aimed at evaluating the effects of maternal exposure to potassium cyanide (KCN) during lactation in goats. Twenty-seven lactating female goats were orally dosed with 0 (control), 1.0, 2.0, or 3.0 mg KCN/kg body weight/day from lactation days 0 to 90. After this period, all male kids and one mother from each group were killed for a pathological study. Cyanide treatment promoted the clinical signs of maternal toxicity in the highest KCN group but did not affect body weight. Both cyanide and thiocyanate presented increased levels in both dams and kids from the treated groups. Microscopic lesions, but without alterations on the biochemical panel, were found in the brain, thyroid, liver, and kidneys of both dams and kids from the treated groups. These findings suggest that lactating offspring can be indirectly intoxicated by maternal exposure to cyanide.